Hydroperoxides as inactivators of aromatase: 10 beta-hydroperoxy-4-estrene-3,17-dione, crystal structure and inactivation characteristics.

The crystal structure of 10 beta-hydroperoxy-4-estrene-3,17-dione (10 beta-OOH) was determined, and its inhibition of human placental aromatase was investigated. In the absence of added NADPH, 10 beta-OOH caused a time-dependent loss of aromatase activity (e.g., 50% loss after 90 s with 2.16 microM 10 beta-OOH). Protection against this loss of activity was provided when a substrate, androstenedione, was included in the incubation. Centrifugation and resuspension of the 10 beta-OOH-treated microsomes in fresh buffer failed to restore the activity, but partial recovery could be effected by dithiothreitol. Experiments to detect destruction of aromatase protoheme were done but were inconclusive. In the presence of NADPH, 10 beta-OOH did not cause a time-dependent loss of activity but was instead a competitive inhibitor (Ki = 330 nM) of androstenedione (Km = 21 nM) aromatization. The added NADPH was not utilized for the aromatization of 10 beta-OOH to estrogens, and enhanced reduction of 10 beta-OOH to 10 beta-hydroxy-4-estrene-3,17-dione could not be detected. In addition, microsomes alone were incapable of using 10 beta-OOH to support the aromatization of androstenedione. Cumene hydroperoxide and H2O2 were also investigated as inactivators of aromatase. Losses of activity comparable to those found for 10 beta-OOH could only be observed at 500-1000-fold higher concentrations of these agents, and no protection was provided by either androstenedione or NADPH. Extensive destruction of microsomal protoheme was found with these nonsteroidal agents.